Current Protocols in Cytometry最新文献

The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology, but also in defining cellular responses to drug treatments. Changes in DNA replication during S-phase can provide valuable insights into mechanisms of cell growth, cell cycle kinetics, and cytotoxicity. A common method for detection of cell proliferation is the incorporation of a thymidine analog during DNA synthesis. This chapter presents a pulse labeling method using the thymidine analog, 5-ethynyl-2′-deoxyuridine (EdU), with subsequent detection by click chemistry. EdU detection using click chemistry is bio-orthogonal to most living systems and does not non-specifically label other biomolecules. Live cells are first pulsed with EdU. After antibody labeling cell surface markers, fixation, and permeabilization, the incorporated EdU is covalently labeled using click chemistry thereby identifying proliferating cells. Improvements in click chemistry allow for labeling in the presence of fluorescent proteins and phycobiliproteins without quenching due to copper. Measuring DNA replication during cell cycle progression has cell health applications in flow cytometry, fluorescence microscopy, and high content imaging. This protocol has been developed and optimized for research use only and is not suitable for use in diagnostic procedures. © 2017 by John Wiley & Sons, Inc.

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune system. Despite this heterogeneity, we do not routinely measure immune competence in clinical practice, and there are no consensus assays of healthy immune function. Using mass cytometry (CyTOF), we can simultaneously detect ∼40 markers to identify various cell subsets and determine their function by the expression of cytokines, cytotoxicity, and activation markers. This can help assess ‘immunocompetence’ and facilitate better implementation of immunotherapies, both in specific disease settings and perhaps eventually as a prognostic tool in healthy subjects. Here we introduce the concepts behind this assay and provide a protocol that we have successfully implemented to identify possible predictive biomarkers of immunotherapy outcome. © 2017 by John Wiley & Sons, Inc.

Lasers are the principal light sources for flow cytometers. Virtually all cytometers are equipped with at least one (and often many more) lasers. This unit covers the various types of lasers available and the qualities that make them suitable or unsuitable for use in flow cytometers. Also included is a discussion of future directions, particularly in the area of tunable laser development. Practical tips are provided for building multilaser cytometer systems. © 2018 by John Wiley & Sons, Inc.

Multiplexed single-cell experimental techniques like mass cytometry measure 40 or more features and enable deep characterization of well-known and novel cell populations. However, traditional data analysis techniques rely extensively on human experts or prior knowledge, and novel machine learning algorithms may generate unexpected population groupings. Marker enrichment modeling (MEM) creates quantitative identity labels based on features enriched in a population relative to a reference. While developed for cell type analysis, MEM labels can be generated for a wide range of multidimensional data types, and MEM works effectively with output from expert analysis and diverse machine learning algorithms. MEM is implemented as an R package and includes three steps: (1) calculation of MEM values that quantify each feature's relative enrichment in the population, (2) reporting of MEM labels as a heatmap or as a text label, and (3) quantification of MEM label similarity between populations. The protocols here show MEM analysis using datasets from immunology and oncology. These MEM implementations provide a way to characterize population identity and novelty in the context of computational and expert analyses. © 2018 by John Wiley & Sons, Inc.

A number of methods have been developed to compare single parameter histograms. Some perform a channel-by-channel analysis and others give a single statistic about how the histograms may or may not differ. If they do differ, then the significance of the difference or confidence limit is usually provided. The specific location(s) for the greatest deviations may also be given. Some are more effective at resolving severely overlapping populations and others work poorly when there is any significant overlap. Each method makes certain assumptions about the data. It is important to understand the assumptions being made and to understand the limitations of each method. It is essential to know how to identify when a comparison method will work for a given set of histograms. This unit explores the different methods, and provides a guide for the reader to choose the most appropriate method(s) to use for a specific data set(s). © 2018 by John Wiley & Sons, Inc.

Modern digital microscopy combines the equipment of classical light microscopy with a computerized imaging system. The technique comprises image formation by optics, image registration by a camera, and saving of image data in a computer file. This chapter describes limitations that are particular to each of these processes, including optical resolution, efficiency of image registration, characteristics of image file formats, and data management. Further suggestions are given which serve, in turn, to help construct a set of guidelines aimed at optimization of digital microscopic imaging. © 2018 by John Wiley & Sons, Inc.

Mitochondria are dynamic organelles that perform several vital cellular functions. Requisite for these functions are mitochondrial fusion and fission. Despite the increasing importance of mitochondrial dynamics in a range of cellular processes, there exist limited methods for robust quantification of mitochondrial fission and fusion. Currently, the most widely used method to measure mitochondrial fusion is the polyethylene glycol (PEG) fusion assay. While this assay can provide useful information regarding fusion activity, the reliance on manual selection of rare fusion events is time consuming and may introduce selection bias. By utilizing the image-capture features and colocalization analysis of imaging flow cytometry in combination with the PEG fusion assay, we are able to develop a high-throughput method to detect and quantify mitochondrial fusion activity. © 2017 by John Wiley & Sons, Inc.

The poisoning of Topoisomerase II (Top2) has been found to be useful as a therapeutic strategy for the treatment of several tumors. The mechanism of Top2 poisons involves a drug-mediated stabilization of a Top2-DNA complex, termed Top2 cleavage complex (Top2cc), which maintains a 5′ end of DNA covalently bound to a tyrosine from Top2 through a phosphodiester group. Drug-stabilized Top2cc leads to Top2-linked-DNA breaks, which are believed to mediate their cytotoxicity. Several time-consuming or cell type-limiting assays have been used in the past to study drug-stabilized Top2cc. Here, we describe a flow cytometry-based method that allows a rapid assessment of drug-induced Top2cc, which is suitable for high throughput analysis in almost any kind of human cell. The analyses of the drug-induced Top2cc in the cell cycle context and the possibility to track its removal are additional benefits from this methodology. © 2017 by John Wiley & Sons, Inc.